The Molecular Biology of the Bacilli PDF
Preview The Molecular Biology of the Bacilli
MOLECULAR BIOLOGY An International Series of Monographs and Textbooks Editors: BERNARD HORECKER, NATHAN O. KAPLAN, JULIUS MARMUR, AND HAROLD A. SCHERAGA A complete list of titles in this series appears at the end of this volume. The Molecular Biology of the Bacilli Volume II Edited by DAVID A. DUBNAU Department of Microbiology The Public Health Research Institute of the City of New York, Inc. New York, New York 1985 ACADEMIC PRESS, INC. (Harcourt Brace Jovanovich, Publishers) Orlando San Diego New York London Toronto Montreal Sydney Tokyo COPYRIGHT © 1985, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER. ACADEMIC PRESS, INC. Orlando, Florida 32887 United Kingdom Edition published by ACADEMIC PRESS INC. (LONDON) LTD. 24-28 Oval Road, London NW1 7DX LIBRARY OF CONGRESS CATALOGING IN PUBLICATION DATA Main entry under title: The Molecular biology of the bacilli. (Molecular biology) Includes bibliographies and index. Contents: v. 1. Bacillus subtilis / edited by David A. Dubnau — v. 2. [without special title]. 1. Bacillaceae. 2. Molecular biology. I. Dubnau, David A. II. Series. [DNLM: 1. Bacillus. QW 127.5.B2 M718] QR82.B3M64 1985 589.9'5 81-22815 ISBN 0-12-222702-6 (v. 2 : alk. paper) PRINTED IN THE UNITED STATES OF AMERICA 17 6 5 4 3 2 1 I dedicate this volume to my parents, FANNIE and ISIDORE DUBNAU, in appreciation for their unstinting love and guidance. Contributors Numbers in parentheses indicate the pages on which the authors' contributions begin. Robert Andrews1 (185), Biology Laboratory, Stauffer Chemical Company, Rich- mond, California 94804 Lee A. Bulla, Jr. (185), Department of Biochemistry, University of Wyoming, Laramie, Wyoming 82071 Bruce C. Carlton2 (211), Department of Genetics, University of Georgia, Ath- ens, Georgia 30602 Robert M. Faust (185), Insect Pathology Laboratory, Beltsville Agricultural Research Center, Agricultural Research Service, U.S. Department of Agri- culture, Beltsville, Maryland 20705 Magda H. Gabor (109), Department of Biological Sciences, State University of New York, Albany, New York 12222 José M. Gonzalez, Jr.2 (211), Department of Genetics, University of Georgia, Athens, Georgia 30602 Nelson Goodman (185), Biology Laboratory, Stauffer Chemical Company, Richmond, California 94804 Paul W. Hager (1), Department of Biochemistry, University of California, Berkeley, California 94720 RollinD. Hotchkiss (109), Department of Biological Science, State University of New York, Albany, New York 12222 J. Oliver Lampen (151), Waksman Institute of Microbiology, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854 Peter S. F. Mézes3 (151), Waksman Institute of Microbiology, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854 David O. Nettleton (53), Department of Biochemistry, University of Illinois, Urbana, Illinois 61801 George W. Ordal (53), Department of Biochemistry, University of Illinois, Urbana, Illinois 61801 1 Present address: Department of Microbiology, Iowa State University, Ames, Iowa 50010. 2 Present address: Ecogen, Inc., Princeton, New Jersey 08540. 3 Present address: Dow Chemical Central Research Biotechnology Laboratory, Midland, Michi- gan 48640. ix X CONTRIBUTORS Patrick J. Piggot (73), Division of Microbiology, National Institute for Medical Research, Mill Hill, London NW7 1AA, England Jesse C. Rabinowitz (1), Department of Biochemistry, University of California, Berkeley, California 94720 Ronald E. Yashin (33), Department of Microbiology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 Preface Volume II of The Molecular Biology of the Bacilli combines, as does Volume I, material of interest to molecular biologists concerned with acquiring basic knowledge and to investigators attempting to develop the bacillus system for industrial applications. These concerns are, or should be, inextricably linked. Our progress in developing applications will benefit from our understanding of fundamental problems, and attempts at the former can provide an expanded data base and an important impetus for the latter. The clear relationships between the development of bacilli as insecticides and the biology of sporulation, or between the production of useful cloned products and the biochemistry and genetics of protein secretion, dramatically exemplify this reciprocal and dynamic inter- action. In this volume we have stressed those areas of bacillus research that have recently received attention either because they are unique to bacilli (Chapters 1, 4, 5, 8) or because they present interesting comparisons with other bacteria, primarily Escherichia coli (Chapters 2, 3, 6). I would like to thank Issar Smith and Eugenie Dubnau for advice and encour- agement during the preparation of this volume and Annabel Howard for much of the secretarial assistance. David A. Dubnau Translational Specificity in Bacillus subtilis PAUL W. HAGER AND JESSE C. RABINOWITZ Department of Biochemistry University of California Berkeley, California I. Introduction 1 II. Translational Machinery 3 A. Ribosomes 3 B. Ribosomal Proteins 4 C. Ribosomal RNAs 5 D. Initiation Factors 7 E. Transfer RNAs, Activating Enzymes, and Codon Usage 8 F. Messenger RNAs : 9 III. A Hypothesis for Translational Specificity 13 IV. Test of the "Simple Hypothesis" for Translational Specificity 20 V. Conclusions 26 References 29 I. Introduction Elucidation of the process and components involved in protein synthesis, like so many other biological problems, has depended on the availability of an active cell-free system. Such a system was first described in bacteria for Escherichia coli (Lamborg and Zamecnik, 1960). It was composed of ribonucleoprotein particles, later recognized as ribosomes, a high-speed supernatant fraction, an ATP-generating system, GTP, and Mg2+. Results related to translation in het- erologous systems in which ribosomes and enzymatic factors derived from E. The Molecular Biology Copyright © 1985 by Academic Press, Inc. of the Bacilli All rights of reproduction in any form reserved. ISBN 0-12-222707.-6 1 2 PAUL W. HAGER AND JESSE C. RABINOWITZ coli were used to translate mRNA derived from either bacteria or phages related to bacteria other than E. coli suggested that the mRNA was efficiently translated by the E. coli system (Bassel et al., 1974) and led to the general assumption that components of the protein translational system, including the mRNA, were interchangeable. This point of view was generally accepted because it was found that the cellular components of the protein synthesis machinery were very similar chemically among the bacterial species examined. However, a limited number of observations were reported suggesting that the components of the protein translational apparatus of prokaryotes were not al- together interchangeable. Ribosomes from different bacterial species differed in their ability to translate the same mRNA (Lodish, 1969, 1970a). The ribosomes from E. coli and Bacillus stearothermophilus were found to translate the E. coli phage f2 RNA quite differently. Escherichia coli ribosomes produced unequal levels of three protein products in vitro. (More recently it has been recognized that there are four products.) The most abundant product was the coat protein, followed by the replicase, with the A (maturation) protein made in the least amount. In contrast, the overall incorporation by B. stearothermophilus ribosomes in response to f2 RNA was only —5% of that of E. coli, although the amount of A protein made by the two systems was equal. This difference in selection of translation initiation sites was due to the source of the ribosomes and not to the source of the supernatant fraction or tRNAs used (Lodish, 1969). Lodish (1970a) showed that this selectivity in initiation was dependent on the source of the 30S subunit of the ribosome. The origin of the 50S subunit or initiation factors (present in a salt wash of 70S ribosomes) had no effect. Species-specific translation has also been observed with ribosomes from Clostridium pasteur ianum (Himes et al., 1972). As with/?, stearothermophilus, the C. pasteurianum ribosomes are active on poly(U), but not on f2 RNA. This work showed that C. pasteurianum polyribosomes were translationally active, and that crude mRNA from C. pasteurianum was active in vitro with both E. coli and C. pasteurianum ribosomes. Similar to the result in the B. stearother- mophilus system, C. pasteurianum ribosomes demonstrated species-specific translation (i.e., inability to translate f2 RNA) in the presence of either E. coli or C. pasteurianum initiation factors. In somewhat more general studies, it was found that although ribosomes from E. coli could translate f2 RNA, formaldehyde-treated f2 RNA, T4 early mRNA, E. coli mRNA, and Clostridium pasteurianum mRNA, a ribosome system from C. pasteurianum could translate C. pasteurianum mRNA but not the other four messengers (Stallcup and Rabinowitz, 1973a,b). These studies were extended to other gram-negative (Pseudomonas fluorescens and Azotobacter vinelandii) as well as to gram-positive bacteria (B. subtilis, C. acidi-urici, C. tetanomorphum, Streptococcus faecalis, and Peptococcus aerogenes), with results that suggested that protein synthesis systems derived from E. coli and other gram-negative 1. TRANSLATIONAL SPECIFICITY IN BACILLUS SUBTIUS 3 bacteria were capable of translating mRNA derived from any bacterial source or phage, whereas systems derived from gram-positive organisms could only trans- late mRNA derived from gram-positive organisms (Stallcup et al, 1974, 1976). This phenomenon was referred to as "translational specificity." This specificity was also noted in translational systems derived from B. subtilis, which could translate mRNA derived from B. subtilis or other gram-positive organisms, as well as mRNA from phages related to B. subtilis such as φ29 or SP82, but not mRNA related to coliphage f2 or early T4 RNA (Legault-Demare and Cham- bliss, 1975; Stallcup et al., 1976; Leventhal and Chambliss, 1979). IL Translational Machinery In seeking the molecular basis of the translational specificity observed in prokaryotes, each of the components of the translational machinery could be considered as a potential determinant of that specificity. These components include Ribosomes Ribosomal proteins Ribosomal ribonucleic acids (rRNAs) Initiation factors Transfer RNAs and activating enzymes Messenger RNAs (mRNAs) We shall consider the possible effect of each of these components on transla- tional specificity from a consideration of their specific function in the transla- tional process. Much of this information is based on investigations of the process in E. coli. In a previous chapter in this treatise, Smith (Volume 1, Chapter 4) described the genetic determinants of the translational apparatus in B. subtilis and the regulation of their response to environmental factors. A. Ribosomes Lodish (1970a) demonstrated that the 30S subunit of the ribosome plays the key role in cistron selection in species-specific translation. The identification of the individual components of the 30S subunit responsible for this species-specific translation followed Nomura's pioneering work on the reassembly of the 30S subunit. Nomura et al. (1968) showed that the 30S subunit of ribosomes from E. coli, Micrococcus lysodeikticus, Azotobacter vinelandii, and B. stear other - mophilus could be reconstituted from rRNA and protein fractions (Nomura et al., 1968). In addition, the rRNA and protein fractions could be heterologously mixed to produce 30S particles active in a poly(U)-primed translation assay. These and other single-protein replacement studies (Higo, 1973) indicated the
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